The long-term objective of the proposed research is to determine the effects of age on the susceptibility of the retinal pigment epithelium (RPE) to oxidative stress. Oxidative stress to the RPE is believed to decrease the support that the RPE provides to the adjacent retina, contributing to diseases like age-related macular degeneration. The pigment melanin, which has antioxidant properties, could theoretically be an important RPE antioxidant. However, when melanin is irradiated with visible light it can become a pro-oxidant. It is therefore not clear whether melanin actually protects RPE cells from oxidative damage or does the reverse. Further, within cells melanin is sequestered in a granular organelle, the melanosome, that can restrict interaction between melanin and damaging species. So whatever function melanin performs it is likely limited to the near domain of the granule. Additionally, RPE melanosomes are long-live organelles that could undergo aging changes that alter their redox function. The hypothesis of this proposal is that the melanosome can either decrease or increase oxidative damage to the RPE. Which function it performs is determined by aging, which makes the melanosome more pro-oxidizing due to changes in the physicochemical properties of its major component melanin. In this project a cell biologist and a biophysicist will use innovative strategies developed to measure pro- and antioxidant effects in the immediate domain of pigment granules, both isolated and within RPE cells. `Young'and `aged'RPE melanosomes or control particles will be used in experiments to address the following specific aims: (1) To determine whether and how isolated RPE melanosomes modify the oxidation state of nearby, granule-associated proteins and lipids. Melanosomes will be coated with specific molecules and then subjected to photosensitized and other oxidizing systems under conditions that discriminate different mechanisms of action of melanin. (2) To determine whether and how melanosomes within living RPE cells affect cellular responses to oxidative stress, detected by measures of cell survival, cell spreading or particle motility. Motility depends at least partly on the integrity of the stress-susceptible cytoskeleton and provides a functional measure of events occurring in the sub-cellular domain of the particle. Methods will include electron spin resonance (ESR) spectroscopy, biochemical analyses of protein oxidation and lipid peroxidation, scanning EM and atomic force microscopy of melanosomes treated to simulate aging, replicative senescence protocols to induce cellular aging of the RPE, and novel applications of time-lapse imaging to quantify the responses of living RPE cells to lethal or sub-lethal oxidative stress induced by visible light or chemical oxidants. PUBLIC HEALTH RELEVANCE: RPE cells support the survival of retinal photoreceptors, but as RPE cells age they are believed to function less efficiently due in part to a lifetime of oxidative stress, including stress due to light exposure. RPE cells contain the light-absorbing pigment melanin, which can theoretically also act as both a pro-oxidant and an antioxidant. In this project, the properties of melanin, which is found inside granules called melanosomes, will be studied to determine whether the granules increase or decrease RPE cell susceptibility to oxidative damage. The effects of age on melanosomes will also be studied to determine whether their ability to protect RPE cells from stress declines over time, which could contribute to diseases of the retina such as age-related macular degeneration.